induction and overexpression of a 7 kDa proteins in E coli

Zhonglin Chai via methods%40net.bio.net (by Zhonglin.Chai from bakeridi.edu.au)
Thu Aug 19 17:34:00 EST 2010


Possibility for this small protein to run out of your gel? PAGE  
condition? Do you have other method to detect the presence of your  
protein? ELISA?

Sent from my iPhone

On 20/08/2010, at 5:11 AM, "venkatramanan rao" <ventigers from gmail.com>  
wrote:

> Hello all,
> I want to do domain mapping of my protein of intrest for testing its
> interaction with a  few predicted interactors. I have generated
> N-termius end and C-terminus end of my protein. I have cloned
> approx150 bp both the terminii in pET28a vector between Nde1 and EcoRI
> sites(both the constructs). I tried inducing the protein in BL-21DE3
> cells using but no success. The full length protein expresses normally
> without any problem in a different vector backbone(infact gives a big
> blob of pure protein in induced cells) . I am wondering what went
> wrong. I have sent my  plasmid samples for sequencing and  awaiting
> for results. The constructs were PCR amplified using Pfu
> polymerase(very less chances of errors in PCR product). Since I have
> done directional cloning chances of frameshift is also less. I tried
> for presence of inserts using both colony PCR and plasmid as template
> both methods show presence of template of correct size. I was
> wondering if anybody has expressed protein of similar size. Is it
> getting degraded. Please help me in this regard.
>
> -- 
> Rao Venkatramanan G.
> Research Scholar
> UM-DAE-CBS
> Mumbai University campus,
> Kalina, Santacruz
> Mumbai-400098
>
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